Salomão José Cohin de Pinho
Estudo da viabilidade do cladócero tropical
Macrothrix elegans Sars, 1901 (Cladocera,
Macrothricidae) como organismo-teste em
ensaios ecotoxicológicos.
Salvador 2007
Salomão José Cohin de Pinho
Estudo da viabilidade do cladócero tropical
Macrothrix elegans Sars, 1901 (Cladocera,
Macrothricidae) como organismo-teste em
ensaios ecotoxicológicos.
Dissertação apresentada ao Instituto de Biologia da Universidade Federal da Bahia, para a obtenção de Título de
Mestre em Ecologia e
Biomonitoramento.
Orientador: Prof. Dr. Eduardo Mendes da Silva.
Co-orientadora: Drª. Carla Chastinet
Salvador 2007
BIBLIOTECA CENTRAL REITOR MACÊDO COSTA -UFBA
P654 Pinho, Salomão José Cohin de.
Estudo da viabilidade do cladócero tropical Macrothrix elegans Sars, 1901 (Cladocera, Macrothricidae) como organismo-teste em ensaios ecotoxicológicos / Salomão José Cohin de Pinho. - 2007.
31 f. + anexos.
Orientador: Prof. Dr. Eduardo Mendes da Silva. Co-orientadora: Drª. Carla Chastinet.
Dissertação (mestrado) - Universidade Federal da Bahia, Instituto de Biologia, 2007.
1.Toxicologia ambiental - Testes. 2. Cladócero. 3. Cloreto de potássio. 4. Cloreto de cádmio. I. Silva, Eduardo Mendes da. II. Chastinet, Carla. III. Universidade Federal da Bahia. Instituto de Biologia. IV. Título.
CDD - 577 CDU - 504
Dedico este trabalho à minha esposa, amiga e companheira,
Agradecimentos
Agradeço a Deus por tudo que me ofereceu ao longo da minha vida, à minha família, que sempre se dedicou para que eu chegasse onde estou hoje e em especial aos meus pais, que sempre acreditaram em mim, e apoiando em todos os momentos. À minha esposa pelo carinho atenção e colaboração em todos os momentos, principalmente os mais difíceis.
Ao meu primo Bruno, que sempre foi mais que um irmão e, da maneira dele, me apoiou sempre que precisei.
Agradeço a dedicação e empenho do meu orientador Eduardo Mendes da Silva e da minha co-orientadora Carla Chastinet, ambos tiveram paciência e acreditaram em mim, me dando forças e confiança para que eu terminasse esta dissertação.
À Rejâne Lira pela confiança e força no início do meu curso, sem o qual possivelmente, eu não o teria feito.
À Isabel Lopes e Rui Ribeiro, pelas contribuições únicas durante minha jornada. À Profª. Dra. Ana Lucia Fonseca, por aceitar participar da banca examinadora.
Ao Cristiano, grande amigo que fiz durante este período e que até hoje colabora para que eu siga bem meu caminho, um agradecimento muito especial.
A todos do Marenba, Bruno Abdon, Júlia Niemeyer, Kátia e todos que por ele passaram e de alguma forma deram sua contribuição.
À Sheila Bonfim, que se revelou confiável e dedicada, tendo dado grande contribuição ao meu trabalho.
À Alice, que desde que cheguei ao grupo Marenba, me acolheu e ajudou sempre que precisei.
À Jorgelina Costa, por ter me apoiado nos momentos de dúvida em relação às análises físico-químicas, trazendo segurança.
À Jussara, secretária do programa de Pós-Graduação que “me salvou” diversas vezes. Aos meus colegas e professores do Mestrado, pela companhia agradável durante o curso, em especial a “equipe água”.
E por fim, aqueles que aqui não citei, mas que foram muito importantes para que tudo isto fosse possível, meu muito obrigado.
Sumário
Introdução geral 1
Conclusão geral 3
Referências Bibliográficas 4
Apêndice: MACROTHRIX ELEGANS SARS, 1901 (CLADOCERA, MACROTHRICIDAE): A VIABLE ALTERNATIVE FOR ECOTOXICOLOGICAL
TESTS IN TROPICAL FRESHWATER
6
Introdução geral
1 2
A água é fundamental para o desenvolvimento humano, entretanto, a sua qualidade pode ser
3
influenciada por muitas das atividades humanas (industrial, agro-pastoril, urbanização),
4
perturbando a condição biológica de ecossistemas aquáticos e sendo prejudicial para a saúde
5
humana (Primack & Rodrigues, 2002; Durusoy & Kambur, 2003). A degradação na qualidade da
6
água gera incertezas em relação ao custo-benefício desses avanços, devido à criação de novas
7
substâncias químicas lançadas no ambiente a cada dia, sem sequer saber quais efeitos essas
8
substâncias terão no ambiente e no ser humano (Esteves, 1998; Espíndola et al., 2000; Knie &
9
Lopes, 2004).
10 11
O desenvolvimento da sociedade atual veio seguido de uma série de dependências, trazendo, em
12
conjunto, efeitos diretos e indiretos ao meio ambiente e ao próprio ser humano, muitos deles
13
irreversíveis, em todo o planeta (Moss,1980; Esteves, 1998; Espíndola et al., 2000; Chastinet,
14
2002; Knie & Lopes, 2004).
15 16
Algumas das formas mais comuns de degradação deste ecossistema são os despejos de efluentes
17
em rios, lançamento de esgoto tratado de forma ineficiente, dragagem, construções de
18
hidrelétricas e a mineração, que causa a liberação de metais para o ambiente (Lewis et al., 1999).
19 20
Cada vez mais, o homem sofre as conseqüências do uso inadequado dos recursos naturais, sendo
21
que um dos mais preocupantes atualmente é a contaminação da água por metais, que além de
22
causar graves doenças, também afeta de forma geral os organismos aquáticos, reduzindo a
23
capacidade dessas comunidades de se reproduzir e manter a espécie no local, gerando um grave
24
desequilíbrio (Krane,1999; Brooks, 2004).
1
Atualmente, uma ferramenta muito utilizada para avaliar os riscos e perigos que o ambiente corre
2
é a análises de risco ecológico (ARE) (CEO, 2003), onde a integração dessa ARE à avaliação de
3
risco à saúde humana segue como a abordagem mais atual e poderosa (Pereira et al., 2004). Esta
4
avaliação ocorre em três etapas, (i) a identificação e formulação do problema que gera dois
5
produtos (i.i) determinação dos endpoints (alvos fisiológicos) e (i.ii) modelos conceituais), (ii) a
6
fase de análise e (iii) a fase de caracterização do risco (U.S. EPA, 1998).
7 8
A utilização de espécies de alta relevância ecológica, representatividade e função ecológica, para
9
esses ecossistemas, possibilita à Ecotoxicologia tropical maior autonomia, deixando de ser
10
apenas uma extensão da Ecotoxicologia desenvolvida nos países temperados, visto que são
11
ecossistemas com características bem diferenciadas (Ricklefs, 2003; Zagatto & Bertoletti 2006).
12 13
Atualmente, existe uma grande preocupação em relação a estes impactos no ambiente aquático,
14
fazendo com que uma série de medidas sejam tomadas, dentre elas resoluções
15
(CONAMA357/05, por exemplo) e normas visando proteger e preservar este ambiente
16
(Espíndola et al., 2000; Knie & Lopes, 2004). No entanto, para as regiões de clima tropical, em
17
termos de ensaios ecotoxicológicos, ainda existe uma dependência muito grande dos países de
18
clima temperado, principalmente no Brasil, fazendo uso de espécies exóticas, reduzindo a
19
relevância ecológica dos resultados obtidos (Zagatto & Bertoletti 2006). Sendo assim, é de suma
20
importância o estudo de organismos que sejam representativos e endêmicos de regiões com
21
clima tropical. O presente trabalho visa avaliar a viabilidade do cladócero tropical Macrothrix
22
elegans como potencial organismo-teste em ensaios ecotoxicológicos em ambientes tropicais. 23
Conclusão geral
1 2
Após todos os experimentos com as substâncias de referência, M. elegans demonstrou ser
3
sensível a ambas, apresentando um coeficiente de variação baixo, estando este apto a ser
4
utilizado como organismo-teste para ambientes contaminados por cádmio.
5 6
M. elegans demonstrou ter um grande potencial para ser utilizado em ensaios ecotoxicológicos 7
dos mais variados tipos, mesmo quando comparado outros cladóceros com ensaios já
8
padronizados. No entanto, ainda é necessário que sejam realizados mais investigações para que
9
este cladócero possa ser incluído em normas de padronização.
10 11
Referências bibliográficas
1 2
Brooks, B.W., Stanley, J.K., White, J.C., Turner P.K., Wu, K.B. & La Point, T.W., 2004.
3
Laboratory and field responses to cadmium: an experimental study in effluent-dominated
4
stream mesocosms. Environmental Toxicology and Chemistry, 23 (4), 1057–1064.
5 6
Chastinet, C. A. Z., 2002. Utilização de ovos de Daphnia.magna em ensaios ecotoxicológicos
7
Doctorate theory in Ecology. University of Coimbra.
8 9
Durusoy, M., Kambur, S., 2003. The Application of the Umu Test System for Screening
10
Mutagenicity of Surface Water. Turkish Journal of Biochemistry 28 (1); 3-7.
11 12
Espíndola, E.L.G, Botta-Paschoal, C.M.R., Rocha, O., Bohrer, M.B.C., Oliveira-Neto, A.L. de,
13
2000. Ecotoxicologia: perspectivas para o século XXI. São Carlos, RiMa.
14 15
Esteves, F. de A., 1998. Fundamentos de limnologia. 2ª Ed. Rio de Janeiro: Interciência.
16 17
Knie, J.L.W., Lopes, E.W.B., 2004. Testes Ecotoxicológicos: Métodos, Técnicas e Aplicações.
18
FATMA/GTZ.
19 20
Krane, D.E., Sternberg, D.C., Burton, G.A., 1999. Randomly amplified polymorphic DNA
21
profile-based measures of genetic diversity in crayfish correlated with environmental
22
impacts. Environ. Toxicol. and Chem. 18 (3), 504–508.
23 24
Lewis, M.A., Mayer, F.l., Powell, R.l., Nelson, M.K., Klaine, S.J., Henry, M.G., Dickson, G.W.,
1
1999. Ecotoxicology and risk assesment for wetlands. Ed. setac press.
2 3
Moss, B., 1980. Ecology of Fresh Waters. Backwell Scientific Publications.
4 5
Pereira, R., R. Ribeiro, F. Gonçalves, 2004. Plan for an integrated human and environmental risk
6
assessment in S. Domingos Mine area (Portugal). Journal of Human and Ecological Risk
7
Assessment. 10 (3) 543-578.
8 9
Primack, B.R., Rodrigues, E., 2002. Biologia da Conservação. Ed. Vida.
10 11
U.S. EPA United State Environmental Protection Agency, 1998. Guildlines for ecological risk
12
assessment.
13 14
Zagatto, A. P., Bertoletti, E., 2006. Ecotoxicologia aquática: princípios e aplicações. Rima, São
15
Carlos.
16 17 18
MACROTHRIX ELEGANS SARS, 1901 (CLADOCERA, MACROTHRICIDAE): A
1
VIABLE ALTERNATIVE FOR ECOTOXICOLOGICAL TESTS IN TROPICAL
2
FRESHWATER
3
SALOMÃO J.COHIN-DE-PINHO,*† CARLA B.A.CHASTINET,†SHEILA J.BONFIM,† RUI RIBEIRO,‡ ISABEL LOPES,‡ 4
LILIANA SARO,‡ ALICE A.M.S.ANDRADE,†BRUNO ABDON,†EDUARDO M. DA SILVA†
5
† Instituto de Biologia da Universidade Federal da Bahia, Campus Universitário, CEP
40170-6
115 Salvador, BA, Brazil
7
‡Instituto do Ambiente e Vida, Departamento de Zoologia da Universidade de Coimbra, Largo
8
Marquês de Pombal, 3004-517 Coimbra, Portugal
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
*Corresponding author: Tel.: +55 71 3263-6525; fax: +55 71 3245-3126
34
E-mail address: salpinho@gmail.com (S.J. Cohin-de-Pinho)
35 36
Abstract
1 2
The current study investigates the suitability of a tropical cladoceran species, Macrothrix elegans
3
Sars (1901) for toxicity testing in ecotoxicological tests at 250C involving two reference
4
substances, cadmium chloride (CdCl2) both in natural and artificial media, and potassium
5
chloride (KCl) in natural medium. The work provides a comparison among other cladoceran
6
species from tropical and temperate environments, and discusses the potential of M. elegans as a
7
bioindicator of stress in tropical waters. An experiment on the animal’s life-span has been
8
included to assess its reproductive potential. On average, at 250C, M. elegans lives 38.2 ± 4.4
9
days and each female produces 211.8±30.4 neonates. The criterion of toxicity adopted in the
10
bioassays was mortality/immobility using neonates up to 24-h old. The CdCl2 48-h EC50 mean
11
values and their confidence intervals were 0.035 mg.l-1 [0.020-0.050 mg.l-1] in natural medium,
12
and 0.018 mg.l-1 [0.012-0.024 mg.l-1] in the artificial medium. The KCl 48-h EC50 mean value
13
(natural medium only) was 749.08 mg.l-1 [625.32-872.76 mg.l-1]. The work demonstrates the
14
great potential of M. elegans as a test organism in ecotoxicological tests involving these
15
substances in tropical freshwater ecosystems.
16 17
KEY WORDS: Cladoceran; Life-table; Ecotoxicological tests; EC50; Cadmium chloride; Potassium
18 chloride. 19 20 21 22
1- INTRODUCTION
1 2
Aquatic ecosystems present high biodiversity in comparison to terrestrial ecosystems (Ricklefs,
3
2004), however due to the attraction human settlements feel for water resources, aquatic
4
ecosystems are severely endangered, as far as biodiversity is concerned (Moss,1980; Esteves,
5
1998; Espíndola et al., 2000; Chastinet, 2002; Knie & Lopes, 2004). Loss of biodiversity is
6
mainly caused by habitat reduction, alteration or destruction, introduction of foreign species and
7
pollution (Primack & Rodrigues, 2002; Ricklefs, 2004). As a result of industrial discharges in
8
water bodies, many aquatic organisms are frequently exposed to complex mixtures (Brooks et
9
al., 2004), sometimes of unknown composition (Knie & Lopes, 2004), that may reduce the
10
natural ability of populations to remain adaptable and productive in their ecosystems (Krane et
11
al., 1999; da Silva et al., 2000).
12 13
A variety of test organisms and endpoints have been used to assess the relative toxicity of a
14
certain pollutant or mixture (Rand et al., 1995). Mortality is usually the most applied response
15
criterion and acute tests are still of great importance for legal reasons to control the emission of
16
toxic substances in the environment (Moss, 1980; Abel & Axiak, 1991), in spite of its low
17
ecological relevance (Gray, 1989; Lacher Jr. & Goldstein, 1997). Invertebrates have been largely
18
used in the assessment of pollutant-induced stress in aquatic ecosystems (Oliveira-Neto &
Botta-19
Paschoal, 2002; Sarma et al., 2005). In addition to their biological and genetic characteristics and
20
pivotal position in the food chain, these organisms are also sensitive to a variety of pollutants
21
normally found in aquatic environments (Lagadic & Caquet 1998; Kast-Hutchenson et al., 2001;
22
Chastinet, 2002). Among aquatic invertebrates, cladocerans play an important role in the
23
ecotoxicological assessment of chemicals in freshwater ecosystems, as they are easily
24
maintained in laboratory at low costs, showing also a low genetic variability in laboratory
cultures (parthenogenesis), and are susceptible to a variety of toxic chemicals (Buikema &
1
Cairns, 1980; Maciorowski & Clarke, 1980). Moreover, cladocerans represent an important link
2
for the transfer of energy between primary producers and the secondary and tertiary consumers
3
(Moss, 1980; Sarma et al., 2005). According to Gray (1989) and Chapman (2002b) the choice of
4
an organism for using in ecotoxicologial tests should be based on ecological parameters,
5
considering that different species may exhibit different levels of tolerance to a pollutant, and that
6
tolerance may also vary among individuals of the same species. Ecologically speaking, this is of
7
extreme importance, as several ecotoxicological tests using aquatic temperate organisms, such as
8
Daphnia magna and Ceriodaphnia dubia are largely used in the tropics. (CETESB, 1998; 9
ABNT, 2005; Beatrici, et al., 2006).
10 11
Tropical and temperate ecosystems differ in many aspects. Generally, tropical ecosystems
12
present specific challenges for research and conservation due to their inherent ecological
13
characteristics (Lacher & Goldstein, 1997). Variations in parameters such as luminosity,
14
temperature, food quality, competition and predation, together with the complex trophic
15
relationships among organisms (Ricklefs, 2004) are essentially important to understand the
16
natural restrictions of ecotoxicological tests in tropical aquatic ecosystems. The strategies of life
17
adopted by tropical and temperate cladocerans suffer direct influence of biotic factors (predation,
18
intra- and inter- specific competition) and abiotic (temperature, luminosity, and oxygen
19
saturation), although temperature seems to be the most significant factor (Sarma, 2005). Despite
20
the tropics shelter about 75% of the biodiversity on the planet, and are under process of
21
economical, social, urban, and industrial expansion thus, naturally requiring more ecological
22
attention, the largest amount of ecotoxicological tests, however, have been carried out in
23
temperate climate (Lacher Jr. & Goldstein, 1997). In this way, it may be unsuitable to apply a
24
standardised bioassay with an exotic species in detriment of finding a local and sensitive species,
which would certainly provide more realistic and ecologically relevant results (Gray, 1989).
1
Therefore, it is necessary the standardisation of ecotoxicological tests using adequate tropical
2
organisms (Gray, 1989; Zagatto & Bertoletti 2006). On the other hand, legal instruments require
3
the attainment of ecotoxicological tests standardised by institutions internationally recognised. In
4
this sense, there is a great demand towards the development of tests involving tropical
5
organisms.
6 7
The present work examines the biology, laboratory cultures, and the suitability of the tropical
8
cladoceran Macrothrix elegans Sars (1901) in ecotoxicological tests. The study aims to
9
determine the potential of this animal as a test organism investigating its sensitiveness to two
10
reference substances (cadmium chloride and potassium chloride) tested in natural and artificial
11
media, thus demonstrating its viability as a test-organism of pollution in tropical freshwater
12
ecosystems.
13 14
2- MATERIALS AND METHODS
1 2
M. elegans was collected from a dam at the Capivari Creek (12°38'24”S; 39°04'25”W) in a 3
district of Cruz das Almas (Ba, Brasil). The animals have been cultured in the laboratory since
4
year 2000 and maintained in water from the Capivari Creek (Andrade, 2003) until 2004.
5
Thereafter, M. elegans has been cultured in water from the Jauá Lake (12°49'12”S; 38°13'11”W)
6
located in (Camaçari, Ba) which has been used as a reference water in other studies (e.g. da Silva
7
et al., 2000; Abdon, 2005; Araújo et al., 2006) thus, justifying its choice. The water was 8
collected monthly, left in the dark to age for about one month, and filtered through a 10-µm net
9
before use.
10 11
In the laboratory, M. elegans organisms were maintained in 600-ml glass vessels containing 450
12
ml of medium. The temperature was set at 25°C±1°C along with a photoperiod of 16:8 h (light:
13
dark). Approximately 50 individuals were placed in each vessel, feeding (daily) on an algae
14
(Pseudokirchneriella subcaptata) suspension with a final density of 106 cells ml-1.day-1. Filtered
15
water from Jauá Lake (no adjustments) was used as natural medium. The ASTM hard water
16
medium (ASTM, 1980) was diluted at 50% in ultra pure water (USFilter) to obtain the artificial
17
medium (1:1). Once a day, all neonates were removed from each culture flask, until the sixth
18
brood, when the adults were then discarded, and new cultures of M. elegans started from
19
neonates of the sixth generation using the procedure of Chastinet (2002) slightly modified. Every
20
three-day, all animals were transferred to a clean flask holding fresh medium and algal
21
suspension at the pre-established density of cells.
22 23
A life-table experiment was carried out to examine biological and reproductive aspects of M.
24
elegans (Shrivastava et al., 1999). A single neonate (up to 24-h old) was placed individually in 25
each of the 11 150-ml glass jars each holding 80 ml of the natural medium (filtered water from
1
Jauá Lake). The number of neonates produced per adult day-1 was counted, and the longevity of
2
each animal was recorded. These data was then used to build a life-table. The animals were then
3
submitted to the same experimental conditions of temperature, photoperiod, and feeding
4
described above for the stock cultures. The neonates were removed and counted daily, and the
5
number of broods per individual recorded. The trial was ended after 44 days following the death
6
of the last M. elegans individual.
7 8
Ultra pure water (USFilter) was used for dilution throughout the experiments, excepting for
9
those involving natural medium. The Daphnia similis standard bioassay (ABNT, 1993) and the
10
work by Araújo (2005) with some modifications were used as reference in all ecotoxicological
11
tests and only animals up to 24-h old were used for testing. A CdCl2-stock solution at 80 mg l-1
12
and 10 g l-1 for KCl was prepared and kept at 4ºC until required. All tests were carried out in
50-13
ml glass flasks containing 40 ml of the test solution. In all 11 tests, four replicates, each holding
14
five animals were used per each of the six concentrations tested per assay. Experimental controls
15
(four replicates each) were set to account for the quality of the natural and artificial media. All
16
tests were performed with neonates of daily controlled broods obtained from the stock cultures.
17
The cadmium 48-h EC50 for M. elegans was determined in eleven tests for animals tested in
18
natural medium (N=11) and in seven tests for animals in the artificial medium (N=7). The KCl
19
48-h EC50 was determined in nine tests only in natural medium (N=9). The pH and conductivity
20
in the test vessels were measured at the beginning of each assay. Mortality/immobility was the
21
endpoint adopted in all tests and under these conditions, the dissolved oxygen was not a limiting
22
factor for testing (Abdon, 2005). The tests lasted for a 48-h period when the number of living
23
organisms were recorded. Animals immobilised for a period of at least 15 consecutive seconds
24
were assumed as dead.
1
EC50 mean values and their 95% confidence limits were calculated using the Probit analysis.
2
Significant statistic differences among CdCl2 and KCl 48-h EC50 values obtained in this study
3
for M. elegans and values for other organisms in comparison with M. elegans, were determined
4
applying the Student t-test (Zar, 1996). The coefficients of variation were also calculated for the
5
reference substances to evaluate the precision level of the results (Zagatto & Bertoletti, 2006).
6 7
3- RESULTS AND DISCUSSION
1 2
Tests aiming to define adequate species of cladocerans to assess pollution in tropical freshwater
3
ecosystems are still in a crescent demand. At the moment, only a test with Ceriodaphnia
4
silvestrii has been included in the Brazilian norm (NBR 13713, 2005). 5
6
M. elegans is a small tropical cladoceran which the total body length ranges from 605 to 1015 7
µm (Kotov, 2004). Hence, taking into account the epibenthic behaviour of M. elegans, these
8
animals may be potentially used in a larger range of ecotoxicological tests than daphnids, for
9
example, which the habits are naturally restricted to the water column. In general, these animals
10
are associated with the macrophytes of the margins of lakes and rivers, occurring sporadically as
11
planktonic (Elmoor-Loureiro, 1997). At laboratory conditions, M. elegans presented in natural
12
medium an average life-span of 38.2±4.4 days with a maximum longevity of 44 days, and
13
primiparous between the 5th and 6th day of life (Figure 1). On average, each female produced
14
211.8 ± 30.4 neonates, and the maximum number of neonates per female was 262 thus, showing
15
the high reproductive capacity of M. elegans in 44 days. In addition, this tropical cladoceran
16
presents: (i) parthenogenetic reproduction allowing small genetic variation among individuals,
17
(ii) large number of neonates per brood, (iii) short life cycle, (iv) the cultures demand small
18
areas, and (v) the animals are easily maintained in the laboratory at low costs. These
19
characteristics are usually some of the requirements for the use of particular specie in
20
ecotoxicological tests (Buikema & Cairns, 1980; Gray, 1989).
21 22
0 2 4 6 8 10 12 14 1 6 11 16 21 26 31 36 41 Period (days) p ro d u c ti o n o f th e n e o n a tes / fe m a le 1
Figura 1: Average of neonates production for female along a life cycle of M. elegans in natural
2
medium.
3 4
M. elegans is a common neotropical species, widely distributed from Argentina to Mexico, 5
normally found in lakes and ponds (Kotov et al., 2004). Araújo (2005) employed M. elegans to
6
monitor a metal-contaminated acidified pond (pH 3.1) in a district of Camaçari (Ba, Brazil),
7
comparing with results from fingerlings of Poecilia reticulata Peters (1859). The cladoceran
8
bioassay produced excellent results and there was no significant statistic difference between the
9
sensitivity levels obtained for P. reticulata and M. elegans. Saro et al. (2006) studied the same
10
pond carrying out an in situ microcosm test to compare the re-colonisation ability of M. elegans
11
in relation to three other tropical cladocerans (Ceriodaphnia silvestrii, C. cornuta and Latonopsis
12
australis). The authors verified that M. elegans was able to re-colonise the studied area in a more 13
efficient way than the other three species, thus showing its ability as a suitable biomonitor. 14
15
The mean value obtained for the cadmium 48-h EC50 test with M. elegans in natural medium was
16
0.035 mg.l-1 [0.020-0.050 mg.l-1] (Figure 2). The pH and conductivity varied from 6.45 to 7.25
17
and from 100 to 250 µS.cm-1, respectively. In general, the water quality of a natural medium is
more susceptible to alterations than that of an artificial medium, which is produced and
1
maintained under laboratory controlled conditions. Therefore, the use of natural media in
2
ecotoxicological tests should be carefully thought about (Knie & Lopes, 2004). Nevertheless, the
3
coefficient of variation in the tests using natural medium was only 15.4%, demonstrating a
4
negligible interference of the medium and a good reproducibility of results. Abdon (2005) used
5
the same natural medium, laboratory conditions, and general procedures to assess the toxicity of
6
cadmium (CdCl2) to the tropical cladoceran C. cornuta obtaining a 48-h EC50 value at 0.103
7 mg.l-1 [0.030-0.141 mg.l-1]. 8 9 0,015 0,020 0,025 0,030 0,035 0,040 0,045 0,050 0,055 0,060 1 2 3 4 5 6 7 8 9 10 11 Tests EC 50 f or C adm ium C hl or ide (m g. l -1) 10
Figure 2: Cadmium toxicity (CdCl2) to neonates of M. elegans exposed in natural medium at
11
25ºC.
12 13
Despite the recommendation of the ISO standard (6341: 1989), that the ASTM medium being
14
used as an appropriate medium for cladoceran cultures, this artificial medium without diluting
15
was not well adequate for the growth of M. elegans. Nevertheless, once diluted in ultra pure
16
water at a ratio (1:1) the animals presented a satisfactory growth. In our toxicity tests, M. elegans
17
revealed a higher sensibility to cadmium when exposed to this metal in artificial medium -
18
cadmium 48-h EC50 at 0.018 mg.l-1 [0.012-0.024 mg.l-1] (Figure 3). The pH and conductivity in
the artificial medium varied from 7.0 to 7.7 and from 220 to 320 µS.cm-1, respectively.
1
According to the results obtained here in artificial medium, M. elegans showed to be more
2
sensitive toward Cd than C. silvestrii, which presented a 48-h LC50 mean of 0.062 mg.l-1
[0.016-3
0.110 mg.l-1] (Oliveira-Neto & Botta-Paschoal, 2002) and D. magna (48-h LC50 at 0.033 mg.l-1
4
(Schuytema et al. 1984). Furthermore, the Cd 48-h LC50 obtained for C. silvestrii was also
5
determined in an artificial medium and was significantly different from that obtained here for M.
6
elegans with a coefficient of variation of only 15.8% (p<0.0001). These results reinforce the 7
great potential of M. elegans as test organism in the assessment of pollutant-induced stress in
8
tropical freshwater ecosystems.
9 10 0,005 0,010 0,015 0,020 0,025 0,030 1 2 3 4 5 6 7 Tests EC 50 fo r Ca d m iu m Ch lo rid e (m g .l -1) 11
Figura 3: Cadmium toxicity (CdCl2) to neonates of M. elegans exposed in artificial medium at
12
250C.
13 14
According to Buikema & Cairns (1980) and Orr et al. (1990) a safe procedure to assure the
15
quality of a certain culture aimed to produce healthy specimens for a reliable experimental
16
control in ecotoxicological tests, consists in testing a reference substance which is expected to
17
affect the organism independently of small variations in the composition of the medium used in
18
the tests. Hence, results obtained by different laboratories following a pre-established
methodology may be more easily compared allowing ecotoxicological tests to be standardised in
1
due time (Soares & Calow, 1993; Knie & Lopes, 2004). Boluda et al., (2002) and
2
Manusadžianas et al., (2003) comment that variation in results of toxicity tests due to chemical
3
interactions (other pollutants or experimental medium) may amplify or reduce the toxic effects of
4
some pollutants, such as the case of chelanting agents, for example, which toxicity may be
5
influenced by dissolved organic matter and water hardness. Knie & Lopes (2004) argue that
6
there is a controversy involving the use of natural and artificial medium in ecotoxicological tests,
7
although it seems agreeable that both are important according to specific situations. Artificial
8
medium are of great important for the standardisation of ecotoxicological tests. On the other
9
hand, tests involving natural medium may produce results of great ecological relevance, as they
10
resembles more the environmental reality of the study area.
11 12
Natural and artificial media used in this work presented a difference between their dissolved
13
organic matter content, with larger concentration in the natural media. Conversely, the hardness,
14
measured as CaCO3, is much higher in the artificial medium (80 mg.l-1) than in the natural
15
medium (20 mg.l-1). Our results, regarding the toxicity of cadmium to M. elegans shows that this
16
tropical cladoceran is significantly more susceptible to cadmium in artificial medium (48-h EC50
17
at 0.018 mg l-1) than in natural medium (48-h EC50 at 0.035 mg l-1) (p <0.0001) (Figure 4).
18
Similarly, Penttinen et al. (1998) found that the sensibility of Daphnia magna towards Cd is
19
higher in artificial medium (48-h LC50 at 0.037 mg.l-1) than in natural medium (48-h LC50 at
20
0.058 mg.l-1). A possible explanation for the higher toxicity of cadmium to M. elegans recorded
21
in this work in the artificial medium is the large contents of dissolved organic matter 19.54 µg.l-1
22
(De Santana, 2004) and humid acids in the natural medium, which may have reduced the toxicity
23
of Cd to the animals tested in the natural medium. The presence of organic compounds in the
24
artificial medium was almost negligible. Akkanen & Kukkonen (2001) found a direct
relationship between increasing values of water hardness and reduced levels of complexation of
1
cadmium by dissolved organic matter due to a preference in binding between ions of calcium and
2
cadmium. The water hardness in our toxicity tests was four-fold higher in the artificial medium
3
than in the natural water. Nevertheless, this difference did not seem to be enough to suppress the
4
affinity of the dissolved organic matter and humic acids to the metal which presumably was
5
responsible for the reduction in the bioavailability of cadmium and thus, its toxicity to the
6
cladocerans in the natural medium (Rand et al 1995; Lewis et al. 1999; Kalis et al. 2006).
7 8 0,000 0,010 0,020 0,030 0,040 0,050 Natural Artificial Cultive medium EC 50 fo r Ca d m iu m Ch lo rid e (m g l -1) A B 9
Figura 4: Toxicity of cadmium (CdCl2) to neonates of M. elegans exposed at 250C in natural
10
medium and artificial medium. Mean 48-h EC50 values and their 95% confidence limits (bars).
11 12
The tests to assess the toxicity of potassium chloride to the tropical cladoceran M. elegans were
13
carried out in natural medium only. The mean value obtained for the KCl 48-h EC50 was at
14
749.08 mg.l-1 [625.32–872.76 mg.l-1] with a coefficient of variation of 8.2%, indicating a low
15
variability and high precision of the results (Figure 5). The pH values varied between 6.63 and
16
7.00 while the conductivity ranged from 96 µS.cm-1 in the controls, which showed no mortality,
17
to 1900 µS.cm-1 in the highest concentration tested. Comparing the results obtained in this work
for the toxicity of KCl to M. elegans with toxicity data determined for D. magna (48-h LC50 at
1
660 mg.l-1) and C. dubia (48-h LC50 at 630 mg l-1) (OECD, 2001), it is noticed that the values are
2
reasonably close to each other.
3 4 0,500 0,600 0,700 0,800 0,900 1,000 1 2 3 4 5 6 7 8 9 Tests EC 50 fo r P o tas siu m Ch lo rid e (m g .l -1) 5
Figura 5: Toxicity of potassium (KCl) to neonates of M. elegans exposed in natural medium at
6
250C. Values of 48-h EC50 and their 95% confidence limits (bars).
7 8 9 10
4- CONCLUSIONS
1 2
The study showed clearly that the tropical cladoceran M. elegans is highly sensitive towards Cd
3
either exposed in natural or artificial medium, even when compared with toxicity levels obtained
4
for other tropical or temperate cladocerans in standardised tests. The variation in susceptibility
5
levels observed between the two media tested may be attributed to differences in their
6
composition. The higher organic matter content in the natural medium may have suppressed the
7
cadmium toxicity to M. elegans. The results obtained either in the natural or artificial medium,
8
however, were highly reproducible. M. elegans showed a great advantage as the animals are well
9
adaptable to both, the natural and artificial medium thus, facilitating the standardisation of tests
10
with this tropical cladoceran. M. elegans was also sensitive to potassium chloride and could be
11
used in the quality control of cultures, particularly considering that these animals do not produce
12
toxic residues, thus avoiding risks to researchers and the environment. The epibentthic behaviour
13
of this tropical cladoceran may represent an advantage over other species considering that these
14
animals could be potentially used in toxicity tests for the evaluation of the interface
sediment-15
water. Overall, the tropical cladoceran, M. elegans, displays a great potential as a test organism
16
in ecotoxicological tests. Nevertheless, more studies are required providing data on the
17
characteristics of this animals and its sensibility to other potential toxic substances.
18 19
ACKNOWLEDGEMENTS
20 21
This work was partially funded by Lyondell Inc. The authors are thankful to J. Malboysson for
22
reviewing an early version of the manuscript. This work was also financed by the Brazilian
23
Research Council (CNPq) (Grant Nr. 620151/2004-8). The authors are also thankful to J.
24
Malbouisson for improving the original manuscript.
25 26
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